Mitochondrial free calcium in hepatocytes was estimated by treating rats with sodium butyrate. Metabolism of such short-chain fatty acids in mitochondria produces pyrophosphate and precipitates free calcium. Thin (80nm) cryosections of liver were sectioned using a cryoultramicrotome and transferred to an AEM for microanalysis. Dry wt. concentrations of calcium as well as diffusible ions (Na+, Mg++, K+, Cl-) were determined. Data were quantitated with software running on a Tractor Northern Analysis system and results were analyzed statistically on a Macintosh computer. Results support the view that there is high (approximately lmM) free calcium in the mitochondria.